CN115417837A - Demethylpentane diterpene component in hyssop extract and preparation method and application thereof - Google Patents
Demethylpentane diterpene component in hyssop extract and preparation method and application thereof Download PDFInfo
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- 150000004141 diterpene derivatives Chemical class 0.000 title abstract description 8
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- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 9
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
- C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/32—Oxygen atoms
- C07D307/33—Oxygen atoms in position 2, the oxygen atom being in its keto or unsubstituted enol form
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The invention relates to the technical field of separation and purification of aerial parts of schizonepeta bracteata, and discloses a norpentane diterpenoid component in a hyssop extract as well as a preparation method and application thereof. The invention discloses Nepetabate K for the first time, and the Nepetabate K has a strong anti-inflammatory effect on RAW264.7 cells stimulated by Lipopolysaccharide (LPS), so that the Nepetabate K can be applied to preparation of anti-inflammatory drugs and anti-inflammatory drugs.
Description
Technical Field
The invention relates to the technical field of separation and purification of aerial parts of schizonepeta bracteata, and discloses a norpentane diterpenoid component in a hyssop extract, and a preparation method and application thereof, wherein the norpentane diterpenoid component is called Nepetabylate K for short.
Background
Nepeta brachiata Benth, a Nepeta plant of Nepeta of Labiatae (Labiatae), also named as "ancestor", is prepared from dried whole plant, and has light fragrance, light taste, slight dampness, lung and liver meridian. Mainly distributed in the countries of Pakistan, iran, nepal, etc. Has effects in generating heat, warming lung, relieving asthma, dispelling cold, relieving cough, eliminating dampness, eliminating phlegm, removing sweat, removing toxic substances, relieving inflammation, and relieving swelling, and can be used for treating damp-cold and viscous respiratory diseases. Although modern pharmacological studies have shown that extracts thereof have significant anti-inflammatory activity, there have been fewer related studies to characterize the chemical components thereof. The abietane diterpenoid component contained in the compound has better anti-inflammatory activity in the anti-inflammatory aspect, small adverse reaction, low price and wide source, thereby being taken into the attention of wide Chinese and foreign researchers.
The medicinal effect of the schizonepeta cataria is mainly derived from terpenoids in the schizonepeta cataria, including monoterpenes and diterpenoids, wherein diterpenoids serving as main ingredients have better anti-inflammatory and bacteriostatic activities. Therefore, the method develops and utilizes the abietane diterpenoid monomeric compounds of the schizonepeta bracteata, further excavates the potential medicinal value of the monomeric compounds, determines and characterizes the structure and the physicochemical properties of the monomeric compounds of the schizonepeta bracteata, and has important significance for developing and utilizing the schizonepeta bracteata.
Disclosure of Invention
The invention provides a norpentane diterpenoid component in a hyssop extract, a preparation method and application thereof, overcomes the defects of the prior art, and discloses Nepetate K for the first time, wherein the Nepetate K has a strong anti-inflammatory effect on RAW264.7 cells stimulated by Lipopolysaccharide (LPS), so that the Nepetate K can be used for preparing anti-inflammatory drugs and anti-inflammatory drugs.
One of the technical schemes of the invention is realized by the following measures: a Nepetabate K, the chemical structural formula is:
the following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:
the Nepetabnate K is obtained according to the following method: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
In the first step, 8mL to 12mL of ethanol is added per 1g of the aerial part of Nepeta cataria.
In the fourth step, the eluent for gradient elution of the high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 90:10.
the second technical scheme of the invention is realized by the following measures: a preparation method of Nepetate K is characterized by comprising the following steps: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
The following is further optimization or/and improvement of the second technical scheme of the invention:
in the first step, 8mL to 12mL of ethanol is added per 1g of aerial part of Nepeta cataria.
In the fourth step, the eluent for gradient elution of the high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 90:10.
the third technical scheme of the invention is realized by the following measures: an application of Nepetabate K in preparing the medicines for preventing inflammation.
The fourth technical scheme of the invention is realized by the following measures: an application of Nepetabate K in preparing the anti-inflammatory medicines.
The fifth technical scheme of the invention is realized by the following measures: an application of Nepetabate K in preparing health-care products for preventing and treating inflammation.
The invention discloses Nepetate K for the first time, and the Nepetate K which is generated by ring rearrangement and contains penta-lactone has stronger anti-inflammatory effect on RAW264.7 cells stimulated by Lipopolysaccharide (LPS), so that the Nepetate K can be applied to preparing medicaments for preventing inflammation and anti-inflammatory medicaments.
Drawings
FIG. 1 is a chemical structural diagram of Nepetate K according to the present invention.
FIG. 2 is a HR-MS spectrum of Nepetate K according to the present invention.
FIG. 3 shows Nepetabnate K of the invention 1 H-NMR spectrum.
FIG. 4 shows Nepetabnate K of the invention 13 C-APT spectrum.
FIG. 5 shows the product H of Nepetabnate K according to the invention 1 -H 1 COSY spectrum.
FIG. 6 is a diagram of the HSQC spectrum of Nepetate K of the present invention.
FIG. 7 is an HMBC spectrum of Nepetabate K according to the present invention.
FIG. 8 is a NOESY spectrum of Nepetabrate K according to the present invention.
FIG. 9 is a bar graph showing the toxic effect of Nepetate K of the present invention on RAW264.7 cells.
FIG. 10 is a graph showing the effect of Nepetabate K of the present invention on TNF- α levels in LPS-induced RAW264.7 cells.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention. The various chemical reagents and chemical articles mentioned in the invention are all the chemical reagents and chemical articles which are well known and commonly used in the prior art, unless otherwise specified; the percentages in the invention are mass weight percentages unless otherwise specified; the solution in the present invention is an aqueous solution in which the solvent is water, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution, unless otherwise specified; the normal temperature and room temperature in the present invention generally mean a temperature of 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to the following examples:
example 1: the Nepetabnate K has a chemical structural formula
Example 2: as an optimization of the above embodiment, it was obtained as follows: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
Example 3: as an optimization of the above example, in the first step, 8mL to 12mL of ethanol was added per 1g of aerial part of Nepeta cataria.
Example 4: as an optimization of the above embodiment, in the fourth step, the eluent for gradient elution by high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 90:10.
example 5: the preparation method of the Nepetabate K is carried out according to the following steps: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
Example 6: the Nepetabrate K is applied to preparing the medicine for preventing inflammation.
Example 7: the Nepetabate K is applied to preparing anti-inflammatory drugs.
Example 8: the Nepetabate K is applied to preparing health care products for preventing and treating inflammation.
Example 9: the Nepetabnate K is obtained according to the following method: firstly, crushing and sieving overground parts of 6kg of schizonepeta cataria, adding 40L of ethanol, soaking for 3 hours at room temperature, heating and refluxing for 3 times and 2 hours each time at 50 ℃, combining refluxing extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta cataria total extract; step two, dispersing 437.0g of the schizonepeta cataria total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking 134.8g of petroleum ether part extract, carrying out gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100, 1, 50; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
The Nepetabrate K obtained in example 9 of the present invention was subjected to hydrogen nuclear magnetic resonance (1H-NMR) and carbon nuclear magnetic resonance (C-NMR) 13 C-APT). HR-MS spectrum, 1 H-NMR spectrum, 13 C-APT spectrum, H 1 -H 1 The COSY spectrum, HSQC spectrum, HMBC spectrum and NOESY spectrum are shown in FIGS. 2 to 8.
Fig. 3 and 4 were subjected to map analysis with reference to fig. 2, 5, 6, 7, and 8, and the peaks in fig. 3 and 4 were assigned, and the peak assignments in fig. 3 and 4 are shown in table 1. As can be seen from the data in fig. 3, fig. 4 and table 1, the chemical structural formula of the nepetanate K of the present invention is shown in fig. 1, and the nepetanate K of the present invention is amorphous powder and is easily soluble in chloroform and methanol.
The Nepetabate K of the invention is subjected to an in vitro anti-inflammatory pharmacodynamic experiment, and the in vitro anti-inflammatory pharmacodynamic experiment utilizes an MTT colorimetric method.
Nepetabate K is used as an experimental group, and indomethacin (anti-inflammatory drug) is used as a control group. MTT colorimetric assay was used to examine the effect of nepetamate K on RAW264.7 macrophage viability. The original 264.7 macrophages in logarithmic growth phase were trypsinized to prepare single cell suspensions, seeded at a density of 1X 104 cells per well in 96-well plates and 5% CO 2 Culturing at 37 ℃ in an incubator 24 hours, then discard the supernatant. The blank control group was cultured with 10% FBS containing DMEM, and the drug group was treated with an aqueous solution of Compound 1, with six replicate wells per concentration. 5% CO at 37 ℃ 2 The culture is continued. After 24 hours of incubation, 10. Mu.L of 5mg/mL MTT was added to each well. After 4 hours of culture, the culture medium was removed. Then, 100 μ L of dimethyl sulfoxide (DMSO) was added to each well, followed by shaking for 10 minutes to achieve complete dissolution. The Optical Density (OD) was measured at 492nm using a microplate reader to calculate cell viability.
The bar graph of the toxic effect of nepetafibrate K of the invention on RAW264.7 cells is shown in fig. 9. Fig. 9 shows that nepetafibrate K has a strong anti-inflammatory effect on RAW264.7 cells stimulated by Lipopolysaccharide (LPS).
The effect of Nepetabate K of the present invention on TNF-. Alpha.levels in LPS-induced RAW264.7 cells is shown in FIG. 10. As can be seen from FIG. 10, TNF-. Alpha.secretion was significantly reduced in each administration group as compared with the normal group and the model group.
In conclusion, the invention discloses Nepetabate K for the first time, and the Nepetabate K has a strong anti-inflammatory effect on RAW264.7 cells stimulated by Lipopolysaccharide (LPS), so that the Nepetabate K can be applied to preparation of medicines for preventing inflammation and anti-inflammatory medicines.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
TABLE 1
Claims (10)
1. A Nepetabnate K, characterized by the chemical structural formula:
2. nepetabylate K according to claim 1, characterized in that it is obtained according to the following process: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabtate K at 17.8 minutes.
3. Nepetabate K according to claim 2, characterized in that in the first step 8 to 12mL of ethanol are added per 1g of aerial part of Nepeta cataria.
4. Nepetabnate K according to claim 2 or 3, characterized in that in the fourth step, the eluent for gradient elution by high performance liquid chromatography is a mixture of methanol and water, wherein the volume ratio of methanol to water is 90.
5. A method for preparing nepetanate K according to claim 1, characterized in that it is carried out according to the following steps: firstly, smashing and sieving overground parts of schizonepeta bracteata, adding ethanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, extracting for 1-3 hours each time, combining reflux extracting solutions each time, and recovering and concentrating under reduced pressure to obtain schizonepeta bracteata total extract; secondly, dispersing the schizonepeta tenuifolia total extract into suspension with water, sequentially extracting with petroleum ether, chloroform and ethyl acetate, and concentrating the extract to obtain extracts of all parts; thirdly, taking petroleum ether part extract, and performing gradient elution and separation on the petroleum ether part extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0, 100; and fourthly, purifying and separating the 2 nd fraction after gradient elution of high performance liquid chromatography, collecting eluate, and obtaining Nepetabrate K at 17.8 minutes.
6. The method of producing Nepetabnate K according to claim 5, wherein in the first step, 8mL to 12mL of ethanol is added per 1g of aerial part of Nepeta cataria.
7. The method for preparing Nepetabnate K according to claim 5 or 6, wherein in the fourth step, the eluent for gradient elution of high performance liquid chromatography is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 90:10.
8. use of nepetamate K according to claim 1 or 2 or 3 or 4 for the preparation of a medicament for the prevention of inflammation.
9. Use of nepetamate K according to claim 1 or 2 or 3 or 4 for the preparation of an anti-inflammatory medicament.
10. Use of nepetamate K according to claim 1, 2, 3 or 4 for the preparation of a health product for the prevention and treatment of inflammation.
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| CN113307785A (en) * | 2021-04-26 | 2021-08-27 | 杭州师范大学 | Abietane diterpenoid compound with anti-tumor effect, preparation method, pharmaceutical composition and application |
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| CN108484699A (en) * | 2016-11-15 | 2018-09-04 | 中国海洋大学 | Bipyridyliums alkaloid, preparation method and use |
| US20190322638A1 (en) * | 2016-11-15 | 2019-10-24 | Ocean University Of China | Dipyridyl alkaloid, preparation method therefor and use thereof |
| CN111423310A (en) * | 2020-04-29 | 2020-07-17 | 沈阳药科大学 | Pimarane diterpenoid and preparation method and application thereof |
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Application publication date: 20221202 |